Date of Award
6-3-2025
Degree Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Microbiology and Immunology
First Advisor
Karen Visick
Abstract
Quorum sensing controls numerous processes ranging from production of virulence factors to biofilm formation. Biofilms, communities of bacteria that are attached to one another and/or a surface, are common in nature and, when they form, can produce a quorum of bacteria. One model system to study biofilms is the bacterium Vibrio fischeri, which forms a biofilm that promotes colonization of its symbiotic host. Many factors promote V. fischeri biofilm formation in vitro, including the symbiosis polysaccharide (SYP), cellulose, and the LapV adhesin but, so far, only a handful of studies have probed connections between biofilm formation and quorum sensing. In this work, I was able to further our understanding of quorum sensing’s contribution to biofilm formation, primarily through studying the quorum sensing-dependent transcription factor, LitR, and its downstream biofilm inhibition pathway. I first showed that LitR negatively influences V. fischeri biofilm formation in the context of a biofilm-overproducing strain. To better understand the importance of LitR, I then identified conditions in which the impact of LitR on biofilm formation could be observed in an otherwise wild-type strain. With these conditions, I investigated LitR’s role and the roles of upstream quorum regulators in biofilm phenotypes. In static conditions, LitR and its upstream quorum regulators, including autoinducer synthases LuxS and AinS, contributed to control over biofilms that were SYP-, cellulose-, and LapV-dependent. In shaking liquid conditions, LitR and AinS contributed to control over biofilms that were primarily cellulose- and LapV-dependent. LitR’s contribution to static pellicle formation also held true in an alternate isolate of V. fischeri, KB2B1, indicating that quorum sensing’s control over biofilm formation may be conserved. To understand how LitR affected the production of these biofilm components, I assessed the dependence of the ∆litR mutant biofilm on their known regulators and if LitR controlled the transcription of their genes. Of the syp regulators, LitR was dependent on the histidine kinase RscS and the Hpt domain of SypF for its pellicle phenotype. However, LitR did not substantially affect rscS or sypA transcription. LitR modestly inhibited cellulose transcription in a manner that depended on the transcription factor, VpsR. LitR induced transcription of the c-di-GMP degrading enzyme, PdeV, which is responsible for inhibiting the presence of surface-associated LapV. The gene upstream of pdeV, rpoQ, shares an intergenic region with VF_A1016, a histidine kinase with similarities to a known negative regulator of biofilm production. I showed that VF_A1016 also inhibited biofilm formation and that VF_A1016 was transcriptionally upregulated by LitR. Based on phenotypic assays, LitR appeared to control VF_A1016 and pdeV transcription to inhibit cellulose- and LapV-dependent biofilms, respectively. Chromatin immunoprecipitation sequencing revealed that LitR directly interacts with the rpoQ/VF_A1016 intergenic region to control their expression. Additionally, peaks were identified within the coding regions of other biofilm-relevant genes such as sypO, bcsA, and lapV, suggesting that LitR may bind within these genes to impact their transcription, and, thus, protein production. These findings expand our understanding of LitR and the quorum sensing pathway in the physiology of V. fischeri and illuminate negative control mechanisms that prevent robust biofilm formation by wild-type V. fischeri under laboratory conditions.
Recommended Citation
Fung, Brittany Loi, "Genetic and Biochemical Analyses Reveal LitR's Role in Vibrio fischeri Biofilm Formation" (2025). Dissertations. 4171.
https://ecommons.luc.edu/luc_diss/4171
